In general, biosensors are defined as integrated measuring systems in which biological recognition structures interact with suitable analytes. In such systems, a physico-chemical signal is produced by the biosensor which can be transformed, via a transducer in the steric vicinity, into a measurable value. Such biosensors are in widespread use in clinical diagnostics, environmental analysis, food analysis, process control and fundamental research. In contrast to physical or chemical biosensors, which usually contain a synthetic component, biological biosensors are formed from biomacromolecules such as nucleic acids (DNA/RNA) or proteins; proteins are very good biosensors because they can highly specifically recognize the widest variety of molecules in cells and can bind with high affinity.
Using such biosensors, metal ions, various sugars such as glucose, nucleic acids or certain chemicals can be detected by means of different interactions with the biosensor. Furthermore, biosensors can contribute to analysing biological processes such as, for example, post-translational modifications, protein-protein interactions, enzyme activities as well as the detection of physical cell parameters such as the pH or the concentration of specific metabolites such as chloride ions in living organisms. Depending on the signal to be detected and the transducer, detection can be made in a variety of manners. Thus, biosensors can be detected optically, electrically, electrochemically, thermally or magnetically.
A further highly specific and sensitive possibility of efficiently detecting a signal by means of a biosensor is the transfer of the signal by fluorescence. To this end, recombinant fluorescence reporter proteins have been developed which either (1) interact directly with the substrate or signal to be determined via a specific sensor domain, whereupon the fluorescence domains fluoresce only in their presence or absence, or (2) the fluorescence properties of a fluorescence domain are directly changed by a change in the cell parameters or cell metabolites, such as a variation in pH. Thus, zinc ions can be detected by means of a modified eYFP (insertion of a zinc finger domain), or calcium ions can be detected by inserting calmodulin into the permutated eYFP.
In order to be able to resolve and detect even small variations, for example cell-specific signals, metabolites or proteins, more and more biosensors have been developed which are based on a Förster resonance energy transfer (FRET) system.
In order to be able to observe the fluorescence-resonance phenomenon, two chromophores with coordinated optical properties are required: a so-called donor and an acceptor. The purpose of the donor is the selective absorption of light emitted by a light source and its subsequent re-radiation, which as a rule is shifted to longer wavelengths. The absorption maximum of the acceptor lies in this emission spectrum, so that it can take up the emitted energy. In general, then, the emission of the acceptor indicates an energy transfer from donor to acceptor, which is described as FRET. The efficiency of the FRET is dependent on a number of factors. Thus, the emission band of the donor must have a sufficient overlap with the absorption band of the acceptor. Furthermore, the separation “r” and the orientation of the chromophores with respect to each other play an important role. In order to obtain a sufficiently intensive signal, “r” should be between 10 and 100 Å. Furthermore, the spatial orientation of the dyes is extremely important as regards the intensity of the energy transfer. This is non-radiative and directional and thus can only take place if the transition dipole moments of the donor and acceptor are orientated as parallel as possible to each other. The development of FRET biosensors means that it is now possible to detect or identify complex cell processes, cell parameters or protein interactions in fundamental research and in biotechnology with far more precision, since the dependency of the fluorescence signal on two fluorescence reporters means that the sensitivity of the signal recognition is greatly increased.
Since the investigation of reaction pathways in living systems and also the identification of the molecules involved therein is very difficult, there is a significant need for analytical methods to be developed which can deliver results rapidly and efficiently. Processes such as protein folding, protein interactions with each other or with DNA or RNA, and reactions of enzymes with their respective substrates are in need of a method for the separation-dependent examination of such processes; thus, FRET systems are also of particular interest as biosensors. With the aid of this “molecular tool”, it is possible to follow separation changes on the nm-scale in real time, and so it is particularly suitable for the in vitro and in vivo analysis of biochemical events.
However, the search for FRET biosensors is very labour-intensive, since the emission bands of the donor must have a sufficiently large overlap with the absorption band of the acceptor. As a rule, therefore, comprehensive experimentation has to be carried out for each and every possible FRET pair.
Until now, green fluorescent protein (GFP) and related fluorescence proteins have been used for single biosensors or as FRET pairs or biosensors.
GFP and its colour variants (for example YFP, yellow fluorescent protein) are in widespread use in many areas of fundamental research and in biotechnology: fluorescent proteins are used for the investigation of gene-regulatory mechanisms or to monitor biotechnological processes. Fluorescence reporters may even be used to investigate cellular differentiation processes and to localize the respective target proteins in the cell. They may also be used to monitor folding processes in heterologous proteins in bacterial expression strains.
GFP or related proteins are used in FRET reactions, for example to assay many and varied ions in eukaryotes and also in prokaryotes and in specific cell types such as nerve cells. With the first FRET-based calcium sensor, it was possible to determine the concentration of Ca2+ ions in cells (Baird et al, 1999); in 2006, Marco Mank's group optimized the speed of the fluorescence intensity change and demonstrated this with the Ca2+ biosensor in nerve cells (Mank et al., 2006).
The fact that the FRET signal and thus also the fluorescence intensity reduces with increasing separation of the FRET partners was used to produce a glucose biosensor (Pickup et al. 2004).
Furthermore, it is now possible to use FRET reactions for the local investigation of various redox processes and physical variables in different cell compartments. Thus, in 2010, Yano et al detected and identified physiological and pathogenic redox states, and thus also pH changes, in peroxisomes using the redoxfluor biosensor.
Despite these various possibilities, there are still a lot of questions which have not yet been answered or have not yet been answered with sufficient accuracy using currently known FRET pairs.
Thus, it would be highly advantageous if further FRET pairs could be provided which reacted, or reacted more sensitively than presently known FRET pairs, to changes in cell parameters or metabolites such as, for example, O2 concentration, variations in pH, temperature changes and changes in specific ion concentrations.